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Purpose:Follicular lymphoma (FL) is the most frequent indolent non-Hodgkin lymphoma. Around 20% of patients suffer early disease progression within 24 months (POD24) of diagnosis. This study examined the significance of circulating tumor DNA (ctDNA) in predicting response to therapy and POD24 in patients with FL.Experimental Design:We collected 100 plasma samples, before and during the treatment, from 36 patients with FL prospectively enrolled in 8 Spanish hospitals. They were treated with a chemotherapy-rituximab regimen and followed up for a median of 3.43 years. We performed targeted deep sequencing in cell-free DNA (cfDNA) and tumor genomic DNA from 31 diagnostic biopsy samples.Results:Of the alterations detected in the diagnostic tissue samples, 73% (300/411) were also identified in basal cfDNA. The mean numbers of alterations per basal cfDNA sample in patients who suffered progression of disease within 24 months (POD24-pos) or did not achieve complete response (non-CR) were significantly higher than in POD24-neg or CR patients (unpaired samples t test, P = 0.0001 and 0.001, respectively). Pretreatment ctDNA levels, as haploid genome equivalents per milliliter of plasma, were higher in patients without CR (P = 0.02) and in POD24-pos patients compared with POD24-neg patients (P < 0.001). Dynamic analysis showed that ctDNA levels decreased dramatically after treatment, although the reduction was more significant in patients with CR and POD24-neg patients.Conclusions:Basal ctDNA levels are associated with the risk of early progression and response to treatment in FL. cfDNA monitoring and genotyping during treatment and follow-up predict response to treatment and early progression.

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Background ALK rearrangements are present in 5% of nonsmall cell lung cancer (NSCLC) tumors and identify patients who can benefit from ALK inhibitors. ALK fusions testing using liquid biopsies, although challenging, can expand the therapeutic options for ALK-positive NSCLC patients considerably. RNA inside extracellular vesicles (EVs) is protected from RNases and other environmental factors, constituting a promising source for noninvasive fusion transcript detection. Methods EVs from H3122 and H2228 cell lines, harboring EML4-ALK variant 1 (E13; A20) and variant 3 (E6a/b; A20), respectively, were successfully isolated by sequential centrifugation of cell culture supernatants. EVs were also isolated from plasma samples of 16 ALK-positive NSCLC patients collected before treatment initiation. Results Purified EVs from cell cultures were characterized by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA), and flow cytometry. Western blot and confocal microscopy confirmed the expression of EV-specific markers as well as the expression of EML4-ALK-fusion proteins in EV fractions from H3122 and H2228 cell lines. In addition, RNA from EV fractions derived from cell culture was analyzed by digital PCR (dPCR) and ALK-fusion transcripts were clearly detected. Similarly, plasma-derived EVs were characterized by NTA, flow cytometry, and the ExoView platform, the last showing that EV-specific markers captured EV populations containing ALK-fusion protein. Finally, ALK fusions were identified in 50% (8/16) of plasma EV-enriched fractions by dPCR, confirming the presence of fusion transcripts in EV fractions. Conclusions ALK-fusion transcripts can be detected in EV-enriched fractions. These results set the stage for the development of EV-based noninvasive ALK testing.

Purpose: Follicular lymphoma (FL) is the most frequent indolent non-Hodgkin lymphoma. Around 20% of patients suffer early disease progression within 24 months (POD24) of diagnosis. This study examined the significance of circulating tumor DNA (ctDNA) in predicting response to therapy and POD24 in patients with FL. Experimental Design: We collected 100 plasma samples, before and during the treatment, from 36 patients with FL prospectively enrolled in 8 Spanish hospitals. They were treated with a chemotherapy-rituximab regimen and followed up for a median of 3.43 years. We performed targeted deep sequencing in cell-free DNA (cfDNA) and tumor genomic DNA from 31 diagnostic biopsy samples. Results: Of the alterations detected in the diagnostic tissue samples, 73% (300/411) were also identified in basal cfDNA. The mean numbers of alterations per basal cfDNA sample in patients who suffered progression of disease within 24 months (POD24-pos) or did not achieve complete response (non-CR) were significantly higher than in POD24-neg or CR patients (unpaired samples t test, P = 0.0001 and 0.001, respectively). Pretreatment ctDNA levels, as haploid genome equivalents per milliliter of plasma, were higher in patients without CR (P = 0.02) and in POD24-pos patients compared with POD24-neg patients (P < 0.001). Dynamic analysis showed that ctDNA levels decreased dramatically after treatment, although the reduction was more significant in patients with CR and POD24-neg patients. Conclusions: Basal ctDNA levels are associated with the risk of early progression and response to treatment in FL. cfDNA monitoring and genotyping during treatment and follow-up predict response to treatment and early progression.

Lung cancer is a major public health problem due to its high incidence and mortality rate. The altered metabolism in lung cancer is key for the diagnosis and has implications on both, the prognosis and the response to treatments. Although Cancer-associated fibroblasts (CAFs) are one of the major components of the tumor microenvironment, little is known about their role in lung cancer metabolism. We studied tumor biopsies from a cohort of 12 stage IIIA lung adenocarcinoma patients and saw a positive correlation between the grade of fibrosis and the glycolysis phenotype (Low PGC-1α and High GAPDH/MT-CO1 ratio mRNA levels). These results were confirmed and extended to other metabolism-related genes through the in silico data analysis from 73 stage IIIA lung adenocarcinoma patients available in TCGA. Interestingly, these relationships are not observed with the CAFs marker α-SMA in both cohorts. To characterize the mechanism, in vitro co-culture studies were carried out using two NSCLC cell lines (A549 and H1299 cells) and two different fibroblast cell lines. Our results confirm that a metabolic reprogramming involving ROS and TGF-β signaling occurs in lung cancer cells and fibroblasts independently of α-SMA induction. Under co-culture conditions, Cancer-Associated fibroblasts increase their glycolytic ability. On the other hand, tumor cells increase their mitochondrial function. Moreover, the differential capability among tumor cells to induce this metabolic shift and also the role of the basal fibroblasts Oxphos Phosphorylation (OXPHOS) function modifying this phenomenon could have implications on both, the diagnosis and prognosis of patients. Further knowledge in the mechanism involved may allow the development of new therapies., Work in the authors’ laboratories is supported by ‘‘Instituto de Salud Carlos III’’ PI13/01806 and PIE14/0064 to M.P. A.C-B, received a Spanish Lung Cancer Group fellowship. R.L-B, is supported by Comunidad Autónoma de Madrid “Garantía juvenil” contract.

Diffuse large B-cell lymphoma (DLBCL) is a heterogeneous disease whose prognosis is associated with clinical features, cell-of-origin and genetic aberrations. Recent integrative, multi-omic analyses had led to identifying overlapping genetic DLBCL subtypes. We used targeted massive sequencing to analyze 84 diagnostic samples from a multicenter cohort of patients with DLBCL treated with rituximab-containing therapies and a median follow-up of 6 years. The most frequently mutated genes were IGLL5 (43%), KMT2D (33.3%), CREBBP (28.6%), PIM1 (26.2%), and CARD11 (22.6%). Mutations in CD79B were associated with a higher risk of relapse after treatment, whereas patients with mutations in CD79B, ETS1, and CD58 had a significantly shorter survival. Based on the new genetic DLBCL classifications, we tested and validated a simplified method to classify samples in five genetic subtypes analyzing the mutational status of 26 genes and BCL2 and BCL6 translocations. We propose a two-step genetic DLBCL classifier (2-S), integrating the most significant features from previous algorithms, to classify the samples as N12-S, EZB2-S, MCD2-S, BN22-S, and ST22-S groups. We determined its sensitivity and specificity, compared with the other established algorithms, and evaluated its clinical impact. The results showed that ST22-S is the group with the best clinical outcome and N12-S, the more aggressive one. EZB2-S identified a subgroup with a worse prognosis among GCB-DLBLC cases.

Key Points Gene expression and mutational analysis confirm the differences among the 3 peripheral TCL subclasses: AITL, PTCL-NOS, and PTCL-TFH.The expression of a gene set, including B-cell genes, is an IPI-independent prognostic factor for AITL cases., Visual Abstract, Peripheral T-cell lymphoma (PTCL) is a clinically aggressive disease, with a poor response to therapy and a low overall survival rate of approximately 30% after 5 years. We have analyzed a series of 105 cases with a diagnosis of PTCL using a customized NanoString platform (NanoString Technologies, Seattle, WA) that includes 208 genes associated with T-cell differentiation, oncogenes and tumor suppressor genes, deregulated pathways, and stromal cell subpopulations. A comparative analysis of the various histological types of PTCL (angioimmunoblastic T-cell lymphoma [AITL]; PTCL with T follicular helper [TFH] phenotype; PTCL not otherwise specified [NOS]) showed that specific sets of genes were associated with each of the diagnoses. These included TFH markers, cytotoxic markers, and genes whose expression was a surrogate for specific cellular subpopulations, including follicular dendritic cells, mast cells, and genes belonging to precise survival (NF-κB) and other pathways. Furthermore, the mutational profile was analyzed using a custom panel that targeted 62 genes in 76 cases distributed in AITL, PTCL-TFH, and PTCL-NOS. The main differences among the 3 nodal PTCL classes involved the RHOAG17V mutations (P < .0001), which were approximately twice as frequent in AITL (34.09%) as in PTCL-TFH (16.66%) cases but were not detected in PTCL-NOS. A multivariate analysis identified gene sets that allowed the series of cases to be stratified into different risk groups. This study supports and validates the current division of PTCL into these 3 categories, identifies sets of markers that can be used for a more precise diagnosis, and recognizes the expression of B-cell genes as an IPI-independent prognostic factor for AITL.

Lung cancer has the highest incidence and mortality rate in the world. One of the most promising new cancer therapies in recent years is immunotherapy, which is based on the blockade of immune checkpoints such as programmed cell death protein 1 (PD-1). Exercise training is beneficial to maintain and improve the quality of life of cancer patients, and it might also modulate the anti-tumoral efficiency of some chemotherapeutic agents. However, the potential of exercise combined with immunotherapy as a cancer therapy remains to be elucidated. Here, we examined the effects of exercise on tumor growth and its possible adjuvant effects when combined with anti-PD-1 immunotherapy (nivolumab) in a patient derived xenograft (PDX) model of non-small-cell lung carcinoma (NSCLC).We generated a PDX model using NOD-SCID gamma mice with subcutaneous grafts from tumor tissue of a patient with NSCLC. Animals were randomly assigned to one of four groups: non-exercise + isotype control (n=5), exercise + isotype control (n=5), non-exercise + nivolumab (n=6) or exercise + nivolumab (n=6). The animals undertook an 8- week moderate-intensity training regimen (treadmill aerobic exercise and strength training). Immunotherapy (nivolumab) or an isotype control was administered 2 days/week, for 6 weeks. Several tumor growth and microenvironment parameters were measured after the intervention.Improvements in aerobic capacity and muscle strength (p=0.027 and p=0.005) were noted in exercised animals. Exercise alone reduced the tumor growth rate with respect to non-exercised mice (p=0.050). The double intervention (exercise + nivolumab) increased tumor necrosis and reduced apoptosis with respect to controls (p=0.026; p=0.030). All interventions achieved a reduction in proliferation compared with the control group (p=0.015, p=0.011, and p=0.011). Exercise alone increased myeloid tumor infiltrates (mostly neutrophils) with respect to the nivolumab only group (p=0.018). Finally, Vegf-a expression was higher in the nivolumab groups (in combination or not with exercise) than in exercise + isotype control group (p=0.045 and p=0.047, respectively). No other significant effects were found.Our results would suggest that aerobic and strength training should be studied as an adjuvant to cancer immunotherapy treatment.

Immunotherapies, such as checkpoint blockade of programmed cell death protein-1 (PD-1), have resulted in unprecedented improvements in survival for patients with lung cancer. Nonetheless, not all patients benefit equally and many issues remain unresolved, including the mechanisms of action and the possible effector function of immune cells from non-lymphoid lineages. The purpose of this study was to investigate whether anti-PD-1 immunotherapy acts on malignant tumor cells through mechanisms beyond those related to T lymphocyte involvement. We used a murine patient-derived xenograft (PDX) model of early-stage non–small cell lung carcinoma (NSCLC) devoid of host lymphoid cells, and studied the tumor and immune non-lymphoid responses to immunotherapy with anti-PD-1 alone or in combination with standard chemotherapy (cisplatin). An antitumor effect was observed in animals that received anti-PD-1 treatment, alone or in combination with cisplatin, likely due to a mechanism independent of T lymphocytes. Indeed, anti-PD-1 treatment induced myeloid cell mobilization to the tumor concomitant with the production of exudates compatible with an acute inflammatory reaction mediated by murine polymorphonuclear leukocytes, specifically neutrophils. Thus, while keeping in mind that more research is needed to corroborate our findings, we report preliminary evidence for a previously undescribed immunotherapy mechanism in this model, suggesting a potential cytotoxic action of neutrophils as PD-1 inhibitor effector cells responsible for tumor regression by necrotic extension. Sin financiación 4.380 JCR (2020) Q1, 17/72 Multidisciplinary Sciences 1.240 SJR (2020) Q1, 10/135 Multidisciplinary No data IDR 2020 UEM

Introduction: Nodal peripheral T-cell lymphoma (PTCL) subclassification (Angioimmunoblastic TCL, PTCL-with T Follicular Helper phenotype and PTCL-NOS) and therapeutic targeting is still controversial. Overall survival (OS) is only around 30% after 5 years. Methods: We performed targeted next-generation sequencing of 61 selected genes in 76 PTCL patients. Among the 76cases, 44 were classified as AITL, 18 as PTCL-TFH and 14 as PTCL-NOS. Results: Our analysis revealed a wide variety of genetic variants that possibly drive the development of AITL, PTCL-TFH and/or PTCL-NOS. We identified an average of 4 variants per patient, mainly clustered in genes regulating chromatin conformation (TET2, DNMT3A) and T-cell differentiation (RHOA, MTOR, IDH2, VAV1 and NOTCH1). More frequently mutated genes were TET2 (76,31%) and DNMT3A (27,63%). Multiple mutations were found in TET2, VAV1, RHOA, NOTCH1, MTOR, JAK1, ZEB1, ATM, DNMT3A and ARID1B. Main difference among the three Nodal PTCL classes was the higher frequency of RHOAG17V mutations (p Conclusions: There is a common molecular basis for the three Nodal PTCL with very frequent mutations in genes regulating chromatin conformation associated with mutations in genes governing T-cell differentiation. RHOA G17V mutations were highly significant enriched in AITL. Findings suggesting Clonal Hematopoiesis were found in the three types of nodal PTCL.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous disease with variable prognosis associated with clinical features, cell-of-origin and genetic aberrations. The main problems for DLBCL patients are that a substantial percentage of them (30-40%) are refractory to treatment or relapse and the lack of accurate predictive markers that adequately determine which patients will benefit from immunochemotherapy. Several recent deep-sequencing studies have proposed new genetic subtypes based on the DLBCL genomic profile (Wright et al., 2020; Lacy et al. 2020) and associated with clinical outcome. However, a consensus and validated classification is still needed. The aim was to determine whether genetic alterations of individual genes or genes clustered in pathways are associated with clinical outcome in immunochemotherapy-treated patients. We used targeted massive sequencing to analyze 84 diagnostic samples from a multicentric cohort of patients with DLBCL treated with rituximab-containing therapies with a median follow up of 6 years. A two-step genetic classifier was built with mutation information from 27 genes and BCL2/BCL6 translocations, based on recently proposed genetic subtypes (Wright et al., 2020; Lacy et al. 2020). Logistic regression and Kaplan-Meier analyses for survival and risk of relapse were performed to assess the potential clinical value of the classifier. Moreover, this two-step classifier was validated in an external cohort and its specificity and sensitivity were tested to determine its accuracy in classifying patients compared to the previous approaches. We found that the most frequently mutated genes were IGLL5 (43%), KMT2D (33.3%), CREBBP (28.6%), PIM1 (26.2%), and CARD11 (22.6%). Mutations in CD79B, PRDM1, and NOTCH2 were associated with a higher risk of relapse after treatment, whereas patients with mutations in CD79B, ETS1, PRDM1, and TNFAIP3 had significantly shorter survival. Analyzing the impact of gene mutations on predefined gene sets related to lymphomagenesis revealed that mutations in genes involved in B-cell development, and BCR-PI3K and MAPK-ERK pathways were significantly associated with a higher risk of relapse and/or shorter overall survival. These results confirmed the current landscape of genetic alterations in DLBCL. According to the two-step classifier, we categorized the samples into MCD, BN2, EZB, ST2, and N1 genetic subgroups (Figure). We tested the accuracy of our classification in an external series (UK population-based Haematological Malignancy Research Network cohort [HMRN]) to determine its specificity and sensitivity compared with their own classification (Lacy et al. 2020) and the LymphGen algorithm (Wright et al., 2020). The comparison demonstrated (Wright et al., 2020; Lacy et al. 2020), a specificity and sensitivity higher than 85% for each subtype, except for BN2. This controversy may be explained by the fact that BN2 is less strongly defined than the other subtypes. We then tested their clinical impact and found the EZB and ST2 subtypes to have a better clinical course and a lower risk of relapse. The BN2 group had the worst clinical outcome of our series, unlike other published studies (Figure). These results were validated in the HMRN cohort, in which N1 showed a higher risk of relapse and shorter overall survival, whereas ST2 is the group with the most favorable outcome. Although N1 was not included for clinical outcome analysis in our cohort due to the small number of cases, the validation of our classifier defined N1 as the most aggressive subtype. In summary, we propose and validate a feasible genetic DLBCL classifier based on an optimized panel of genes that unifies previous genetic classification algorithms in such a way as to facilitate its implementation as part of pathology laboratories for routine patient management. This genetic classifier, combined with clinical data and other molecular characteristics, should eventually help develop improved risk models for DLBCL patients, and guide precision therapy. Figure Disclosures Perez Callejo: F. Hoffmann-La Roche: Current Employment, Current equity holder in publicly-traded company.

BCR-JAK2 is an infrequent gene fusion found in chronic/acute, myeloid/lymphoid Philadelphia chromosome-negative leukaemia. In this study, we demonstrated that in vivo expression of BCR-JAK2 in mice induces neoplasia, with fatal consequences. Transplantation of BCR-JAK2 bone marrow progenitors promoted splenomegaly, with megakaryocyte infiltration and elevated leukocytosis of myeloid origin. Analysis of peripheral blood revealed the presence of immature myeloid cells, platelet aggregates and ineffective erythropoiesis. A possible molecular mechanism for these observations involved inhibition of apoptosis by deregulated expression of the anti-apoptotic mediator Bcl-xL and the serine/threonine kinase Pim1. Together, these data provide a suitable in vivo molecular mechanism for leukaemia induction by BCR-JAK2 that validates the use of this model as a relevant preclinical tool for the design of new targeted therapies in Philadelphia chromosome-negative leukaemia involving BCR-JAK2-driven activation of the JAK2 pathway.

Funding: this study was supported by grants from the Asociacion Espanola contra el Cancer (AECC), Ministerio de Economia y Competitividad (MINECO) (SAF2013-47416-R), Instituto Salud Carlos III (ISCIII) – Fondos de Investigacion Sanitaria (RD06/0020/0107, RD012/0036/0060 and FIS11/1759). The Instituto de Investigacion Marques de Valdecilla (IDIVAL) is partly funded by the Sociedad para el Desarrollo Regional de Cantabria (SODERCAN)

Verruciform xanthoma (VX) is an uncommon benign lesion of unclear etiology which has only been reported twice before in the esophagus. We describe a 70-year-old male who presented an exophytic esophageal lesion incidentally found upon endoscopy 2.7 years following radiation therapy for unresectable squamous cell carcinoma of the tracheal carina. Histologically, the lesion showed a papillary surface change with numerous foamy histiocytes within the lamina propia papillae. Xanthoma cells were strongly positive for vimentin and CD68 (KP1). Polymerase chain reaction did not demonstrate human papillomavirus (HPV) infection. Our results indicate that esophageal VX is not an HPV-induced lesion and suggest a causal relationship between VX and radiotherapy, as previously noted. Histological differential diagnosis is discussed and emphasis is placed on obtaining adequate biopsy material for accurate diagnosis.

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the Western world. Pathogenic mechanisms involve multiple external events (such as microenvironmental and antigenic stimuli) and internal events (genetic and epigenetic alterations) that are associated with the transformation, progression and evolution of CLL. CLL is characterized by an accumulation of mature B cells in peripheral blood, bone marrow and lymphoid tissues. Extracellular stimuli play an important role in the development and maintenance of neoplastic cells. B-CLL cells proliferate and activate pathogenic signaling pathways in anatomical structures known as proliferation centers, which are usually more conspicuous in involved lymph nodes.1 Its clinical course is quite heterogeneous, whereby some patients progress rapidly and have short survival, whereas others have a more stable clinical course that may not need treatment for years. This work was supported by grants from the Ministerio de Economía y Competitividad (MINECO) (SAF2013-47416-R) Instituto de Salud Carlos III (ISCIII)- FEDER – MINECO- AES (CP11/00018, PI10/00621, RD012/0036/0060), and Asociación Española contra el Cancer (AECC). MS-B is supported by a Miguel Servet contract from ISCIII-FEDER (CP11/00018). Salary support to SG is provided by CP11/00018, from ISCIII-FEDER. JG-R is supported by a predoctoral grant from the Fundación Investigación Puerta de Hierro. Sí

BCR-JAK2 is an infrequent gene fusion found in chronic/acute, myeloid/lymphoid Philadelphia chromosome-negative leukaemia. In this study, we demonstrated that in vivo expression of BCR-JAK2 in mice induces neoplasia, with fatal consequences. Transplantation of BCR-JAK2 bone marrow progenitors promoted splenomegaly, with megakaryocyte infiltration and elevated leukocytosis of myeloid origin. Analysis of peripheral blood revealed the presence of immature myeloid cells, platelet aggregates and ineffective erythropoiesis. A possible molecular mechanism for these observations involved inhibition of apoptosis by deregulated expression of the anti-apoptotic mediator Bcl-xL and the serine/threonine kinase Pim1. Together, these data provide a suitable in vivo molecular mechanism for leukaemia induction by BCR-JAK2 that validates the use of this model as a relevant preclinical tool for the design of new targeted therapies in Philadelphia chromosome-negative leukaemia involving BCR-JAK2-driven activation of the JAK2 pathway.

Introduction: Aberrant methylation of the 5′ gene promoter regions is an epigenetic phenomenon that is the one of the major mechanism of inactivation of tumor supressor genes. DNA methylation of the promoter region has been described for several genes in various malignant diseases, and each tumour type may have its own pattern of methylation. To determine the methylation status and expression of cell cycle inhibitors genes (p14, p15, p16), repair genes (MGMT and hMLH1) and the apoptosis regulator gene (DAPKinase) and the possible role of this epigenetic phenomenon in tumour progression of plasma cell disorders, we analyzed the methylation profile of MM, MGUS, and plasmacytomas comparing them with their protein expression. Patients and Methods: A total of 51 cases: 30 MM, 13 MGUS, and 8 plasmacytomas (3 Solitary Bone Plasmacytoma, 5 Extramedullary Plasmacytoma) were included in the study. Bone marrow plasma cells were purified using magnetic microbeads labeled with CD138 in samples with MM and MGUS. Methylation-specific polymerase chain reaction (MSP) for p14, p15, p16, MGMT, hMLH1 and DAPKinase was performed. MSP results were matched to protein expression studies by immunohistochemistry for p15, p16, MGMT and hMLH1. Results: The frequency of aberrant methylation among the MM samples was: 50% for p16, 16.7% for p15, 10% for hMLH1, 23.3% for MGMT, 30% for DAPK. In MGUS samples we found 38.5% for p16, 15.4% for p15, 0% for hMLH1, 7.7% for MGMT and 15.4% for DAPK methylation. The frequency of methylation in plasmacytomas was 62.5% for p16, 25% for p15, 0% for hMLH1, 62.5% for MGMT and 50% for DAPK. p14 was unmethylated in all cases (n=51). The correlation between gene methylation status and protein expression was assessed in 17 MM, 11 MGUS, and 8 plasmacytomas and we found that promoter methylation was strongly associated with gene silencing. All the samples methylated had lost the protein expression. In summary these findings demonstrate that aberrant methylation is an important mechanism of gene silencing in plasma cell disorders: 83.3% of MM, 46.1 of MGUS, and 75% of plasmacytomas have at least one hypermethylated gene (figure 1). We also show, that hypermethylation of p16 is a common phenomenon in plasma cell discrasias while there was no methylation of p14. Although the size of sample is small, we found that hMLH1 hypermethylation was found only in MM, while in plasmacytomas hypermethylation of MGMT and DAPK were frequent events. Moreover in survival studies of MM patients, a trend was observed between simultaneous aberrant methylation of hMLH1 and MGMT and poorer survival, but the number of cases studied limits the statistical analysis and the clinical implications of these results. To better define the clinical impact of methylation markers in plasma cell discrasias, it is therefore necessary to analyze a large number of patient samples. Figure 1. Gene methylation profiling of MM(A), MGUS(B), and plasmacytomas(C) using MSPCR Figure 1. Gene methylation profiling of MM(A), MGUS(B), and plasmacytomas(C) using MSPCR